Production of a 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or 7-chloro-3-methyllquinoline-8-carboxylic acid-tolerant plant by expressing an exogenous 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or 7-chloro-3-methyllquinoline-8-carboxylic acid-binding antibody in the plant

ABSTRACT

Process for production of herbicide-tolerant plants by expressing an exogenous herbicide-binding polypeptide in plants or plant organs. The invention furthermore relates to the use of the corresponding nucleic acids which encode a polypeptide, an antibody or parts of an antibody with herbicide-binding properties in transgenic plants, and the thus transformed plant itself.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for the production ofherbicide-tolerant plants by expressing an exogenous herbicide-bindingpolypeptide in plants or plant organs. The invention furthermore relatesto the use of the corresponding nucleic acids which encode apolypeptide, an antibody or parts of an antibody with herbicide-bindingproperties in transgenic plants, and the thus transformed plant itself.

2. Description of the Related Art

It is known that genetic engineering methods allow the specific transferof foreign genes into the genome of a plant. This process is termedtransformation, and the resulting plants transgenic plants. Transgenicplants are currently being employed in various fields of biotechnology.Examples of insect-resistant plants (Vaek et al. Plant Cell 5 (1987),159-169), virus-resistant plants (Powell et al. Science 232 (1986),738-743) and ozone-resistant plants (van Camp et al. BioTech. 12 (1994),165-168). Examples of improved quality characteristics achieved bygenetic engineering are: improved shelf life of fruit (Oeller et al.Science 254 (1991), 437-439), increased starch production in potatotubers (Stark et al. Science 242 (1992), 419), changes in starch (Visseret al. Mol. Gen. Genet. 225 (1991), 289-296) and lipid composition(Voelker et al. Science 257 (1992), 72-74), and production of foreignpolymers (Poirer et al. Science 256 (1992), 520-523).

An important target of work carried out in the field of plant moleculargenetics is the generation of herbicide tolerance. Herbicide toleranceis characterized by an improved compatibility (in terms of type orlevel) of the plant or plant organs with the herbicide applied. This canbe effected in various ways. The known methods are utilization of ametabolic gene, for example the pat gene, in connection with glufosinateresistance (WO 8705629) or a target enzyme which is resistant to theherbicide, such as in the case of enolpyruvyl shikimate-3-phosphatesynthase (WO 9204449), which is resistant to glyphosate, and the use ofa herbicide in cell and tissue culture for the selection of tolerantplant cells and resulting resistant plants, such as described in thecase of acetyl-CoA-carboxylase inhibitors (U.S. Pat. No. 5,162,602, U.S.Pat. No. 5,290,696).

Antibodies are proteins as component of the immune system. A jointfeature of all antibodies is their spatial, globular structure, theconstruction of light and heavy chain and their basic capability ofbinding molecules or parts of a molecular structure with highspecificity (Alberts et al., in: Molekularbiologie der Zelle [MolecularBiology of the Cell], 2nd Edition 1990, VCH Verlag, ISBN 3-527-27983-0,1198-1237). On the basis of these properties, antibodies have beenutilized for a number of tasks. Application can be divided intoapplication of the antibodies within the animal and human organisms inwhich they are produced, that is to say the so-called in-situapplications, and the ex-situ applications, ie. utilization of theantibodies after they have been isolated from the producing cells ororganisms (Whitelam und Cockburn, TIPS Vol.1, 8 (1996), 268-272).

The use of somatic hybrid cell lines (hybridomas) as a source ofantibodies against very specific antigens is based on work carried outby Köhler and Milstein (Nature 256 (1975) 495-97). This process allowsso-called monoclonal antibodies to be produced which have a uniformstructure and which are produced by means of cell fusion. Spleen cellsof an immunized mouse are fused with mouse myeloma cells. This giveshybridoma cells which proliferate infinitely. At the same time, thecells secrete specific antibodies against the antigen with which themouse had been immunized. The spleen cells provide the capability ofantibody production while the myeloma cells contribute the capacity ofunlimited growth and continuous secretion of antibodies. Since eachhybridoma cell, being a clone, is derived from a single B cell, allantibody molecules produced have the same structure, including theantigen binding site. This method has greatly promoted the use ofantibodies since antibodies which have a single, known specificity and ahomogeneous structure are now available in unlimited quantities.Monoclonal antibodies are used widely in immunodiagnostics and astherapeutics.

In recent years, the so-called phage display method has become availablefor the production of antibodies, and here the immune system and thevarious immunizations in the animal are avoided. The affinity andspecificity of the antibody are made to measure in vitro (Winter et al.,Ann. Rev. Immunol. 12 (1994), 433-455; Hoogenboom TIBTech Vol 15 (1997),62-70). Gene segments which contain the sequence which encodes thevariable region of antibodies, ie. the antigen binding site, are fusedwith genes for the coat protein of a bacteriophage. Then, bacteria areinfected with phages which contain such fusion genes. The resultingphage particles are now equipped with coats containing the antibody-likefusion protein, the antibody-binding domain pointing outward. Such aphage display library can now be used for isolating the phage whichcontains the desired antibody fragment and which binds specifically to acertain antigen. Each phage isolated in this manner produces amonoclonal antigen-binding polypeptide which corresponds to a monoclonalantibody. The genes for the antigen binding site, which are unique foreach phage, can be isolated from the phage DNA and employed forconstructing complete antibody genes.

In the field of crop protection, antibodies were utilized in particularas analytical tools ex-situ for the qualitative and quantitativedetection of antigens. This includes the detection of plantconstituents, herbicides or fungicides in drinking water (Sharp et al.(1991) ACS Symp Ser., 446 (Pestic. Residues Food Saf.) 87-95), soilsamples (WO 9423018) or in plants or plant organs, and the utilizationof antibodies as auxiliaries for the purification of bound molecules.

The production of immunoglobulins in plants was first described by Hiattet al., Nature, 342 (1989), 76-78. The spectrum encompasses single-chainantibodies up to multimeric secretory antibodies (J. Ma and Mich Hein,1996, Annuals New York Academy of Sciences, 72-81).

More recent attempts utilize antibodies in-situ for defending plantsagainst pathogens, in particular viral diseases, by expressing, in plantcells, specific antibodies or parts thereof which are directed againstviral coat proteins (Tavladoraki et al., Nature 366 (1993), 469-472;Voss et al., Mol. Breeding 1 (1995), 39-50).

An analogous approach has also been utilized for defending the plantagainst infection by nematodes (Rosso et al., Biochem Biophys Res Com,220 (1996) 255-263). There exist examples for an application inpharmacology where the in-situ expression of antibodies in plants isutilized for oral immunization (Ma et al., Science 268 (1995), 716-719;Mason and Arntzen, Tibtech Vol 13 (1996), 388-392). The body is providedwith antibodies formed by the plant and originating from plants or plantorgans which are suitable for consumption, via the mouth, throat ordigestive tract, which antibodies cause efficient immunoprotection.Moreover, a single-chain antibody against the low-molecular-weight planthormone abscisic acid has already been expressed in plants, and areduced availability of plant hormones, due to binding of abscisic acidin the plant, has been observed (Artsaenko et al., The Plant Journal 8(5) (1995) 754-750).

Chemical control of weeds in agronomically important crops requires theuse of highly selective herbicides. However, in some cases it isdifficult to develop sufficiently selective herbicides which do notcause damage of the plant which provides the yield in any crop. Theintroduction of herbicide-resistant or -tolerant crop plants cancontribute to solving this problem.

The development of herbicide-resistant crop plants by tissue culture orseed mutagenesis and natural selection is limited. Only those plants canbe manipulated via tissue culture techniques where entire plants can beregenerated successfully from cell cultures. Moreover, followingmutagenesis and selection, crop plants may display undesirablecharacteristics which have to be reeliminated by, in some casesrepeated, back-crossing. Also, the introduction of a resistance byperforming crosses would be restricted to plants of the same species.

It is for the abovementioned reasons that the genetic engineeringapproach of isolating a resistance-encoding gene and transferring itinto crop plants in a targeted manner is superior to the traditionalplant breeding method.

To date, the development of herbicide-tolerant or herbicide-resistantcrop plants, by molecular biology methods, requires a knowledge of themechanism of action of the herbicide in the plant and also that geneswhich impart resistance to the herbicide can be found. A large number ofherbicides which are presently utilized commercially act by blocking anenzyme of an essential amino acid, lipid or pigment biosynthesis step.Herbicide tolerance can be generated by altering the genes of theseenzymes in such a way that the herbicide can no longer be bound and byintroducing these altered genes into crop plants. An alternative exampleis to find analogous enzymes in nature, for example in microorganismswhich exhibit a natural resistance to the herbicide. Thisresistance-imparting gene is isolated from such a microorganism,recloned into suitable vectors and subsequently, after successfultransformation, expressed in herbicide-sensitive crop plants (WO96/38567).

BRIEF SUMMARY OF THE INVENTION

It was an object of the present invention to develop a novel, generallyutilizable genetic engineering method for producing herbicide-toleranttransgenic plants.

We have found that this object is achieved, surprisingly, by a processof expressing, in the plants, an exogenous polypeptide, antibody orparts of an antibody with herbicide-binding properties.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates firstly to the production of aherbicide-binding antibody and the cloning of the relevant gene or genefragment.

The first step is to produce a suitable antibody which binds theherbicide. This can be effected, inter alia, by immunizing a vertebrate,in most cases mouse, rat, dog, horse, donkey or goat, with an antigen.The antigen in this case is a fungicidally active compound which isassociated or coupled to a higher-molecular-weight carrier such asbovine serum albumin (BSA), chicken ovalbumin, keyhole limpethemocyanine (KLH) or other carriers, via a functional group. Afterantigen has been applied repeatedly, the immune response is monitoredwith customary methods, and a suitable antiserum is thus isolated.Initially, this approach yields a polyclonal serum which containsantibodies with differing specificities. For the targeted in-situ use,it is necessary to isolate the gene sequence which encodes a single,specific, monoclonal antibody. A variety of routes are available forthis purpose. The first approach exploits the fusion ofantibody-producing cells and cancer cells to give a hybridoma cellculture which continuously produces antibodies and which finally, bysingling the clones obtained, leads to a homogeneous cell line whichproduces a defined monoclonal antibody.

The cDNA for the antibody, or parts of the antibody, viz. the so-calledsingle chain antibody (scFv), is isolated from such a monoclonal cellline. These cDNA sequences can then be cloned into expression cassettesand used for the functional expression in prokaryotic and eukaryoticorganisms, including plants.

Alternatively, it is possible to select antibodies via phage displaylibraries, and these antibodies bind herbicide molecules and convertthem catalytically into a product which has non-fungicidal properties.Methods for raising catalytic antibodies are described in Janda et al.,Science 275 (1997) 945-948, Chemical selection for catalysis incombinatorial Antibody libraries; Catalytic Antibodies, 1991, CibaFoundation Symposium 159, Wiley-Interscience Publication. Cloning thegene of this catalytic antibody and expressing it in a plant may, inprinciple, also lead to a herbicide-resistant plant.

The invention particularly relates to expression cassettes whoseencoding sequence encodes a herbicide-binding polypeptide or afunctional equivalent thereof, and to the use of these expressioncassettes for the production of a herbicide-tolerant plant. The nucleicacid sequence can be, for example, a DNA sequence or a cDNA sequence.Encoding sequences which are suitable for insertion into an expressioncassette according to the invention are, for example, those whichcontain a DNA sequence from a hybridoma cell which encodes a polypeptidewith herbicide-binding properties and thus impart resistance toplant-enzyme inhibitors to the host.

Moreover, the expression cassettes according to the invention containregulatory nucleic acid sequences which govern expression of theencoding sequence in the host cell. In a preferred embodiment, anexpression cassette according to the invention comprises upstream, ie.on the 5′-end of the encoding sequence, a promoter and downstream, ie.on the 3′-end, a polyadenylation signal and, if appropriate, otherregulatory elements which are linked operatively with the in-betweenencoding sequence for the polypeptide with herbicide-binding propertiesand/or transit peptide. Operative linkage is to be understood as meaningthe sequential arrangement of promoter, encoding sequence, terminatorand, if appropriate, other regulatory elements in such a way that eachof the regulatory elements can function as intended when the encodingsequence is expressed. The sequences preferred for operative linkage,but not limited thereto, are targeting sequences for guaranteeingsubcellular localization in the apoplasts, in the plasma membrane, inthe vacuole, in plastids, into the mitochondrium, in the endoplasmaticreticulum (ER), in the nucleus, in liposomes or in other compartmentsand translation enhancers, such as the 5′-leader sequence from thetobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987)8693-8711).

Suitable promotors [sic] of the expression cassette according to theinvention is, in principle, any promoter which is capable of governingthe expression of foreign genes. Promoters which are preferably usedare, in particular, a plant-derived promoter or a promoter originatingfrom a plant virus. Particularly preferred is the CaMV 35S promotor fromthe cauliflower mosaic virus (Franck et al., Cell 21(1980) 285-294).This promoter contains various recognition sequences for transcriptionaleffectors, which, in their totality, lead to permanent and constitutiveexpression of the gene introduced (Benfey et al., EMBO J. 8 (1989)2195-2202).

The expression cassette according to the invention may also comprise achemically inducible promoter by means of which expression of theexogenous polypeptide in the plant can be controlled at a particularpoint in time. Such promoters, for example the PRP1 promotor (Ward etal., Plant.Mol.Biol.22(1993), 361-366), a promoter which is inducible bysalicylic acid (WO 95/1919443), a promoter which is inducible bybenzenesulfonamide (EP 388186), a promoter which is inducible byabscisic acid (EP335528) or a promoter which is inducible by ethanol orcyclohexanone (WO9321334), have been described in the literature and canbe used, among others.

Other promoters which are particularly preferred are those whichguarantee expression in tissues or plant organs in which the herbicidalactivity takes place. Promoters which guarantee leaf-specific expressiondeserve particular mention. Mention must be made of the potato cytosolicFBPase promoter or the potato ST-LSI promotor (Stockhaus et al., EMBO J.8 (1989) 2445-245).

The stable expression of single-chain antibodies, which amounted to upto 0.67% of the total soluble seed protein in the seeds of transgenictobacco plants, was made possible with the aid of a seed-specificpromoter (Fiedler and Conrad, Bio/Technology 10(1995), 1090-1094). Sinceexpression may also be possible in seeds which have been sown or whichare in the process of germination and may be desired for the purposes ofthe present invention, such germination- and seed-specific promoters arealso regulatory elements which are preferred in accordance with theinvention. Thus, the expression cassette according to the invention cantherefore contain, for example, a seed-specific promoter (preferably theUSP or LEB4 promotor), the LEB4 signal peptide, the gene to beexpressed, and an ER retention signal. The construction of the cassetteis shown by way of example in the form of a diagram in FIG. 1 withreference to a single-chain antibody (scFv gene).

An expression cassette according to the invention is produced by fusinga suitable promoter with a suitable polypeptide DNA and, preferably, aDNA which encodes a chloroplast-specific transit peptide and which isinserted between promoter and polypeptide DNA, and a polyadenylationsignal, using customary recombination and cloning techniques as they aredescribed, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook,Molecular Cloning: A Laboratory manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989) and also in T. J. Silhavy, M. L. Bermanand L. W. Enquist, Experiments with Gene Fusions, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. etal., Current Protocols in Molecular Biology, Greene Publishing Assoc.and Wiley-Interscience (1987).

Particularly preferred are sequences which allow targeting into theapoplast, the plastids, the vacuole, into the plasma membrane, themitochondrium, the endoplasmatic reticulum (ER) or, by the absence ofsuitable operative sequences, residence in the compartment of formation,namely the cytosol (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996),285-423). Localization in the ER and the cell wall have proved to beespecially beneficial for quantitative protein accumulation intransgenic plants (Schouten et al. , Plant Mol. Biol. 30 (1996),781-792; Artsaenko et al., Plant J. 8 (1995) 745-750).

The invention also relates to expression cassettes whose encodingsequence encodes a herbicide-binding fusion protein, part of the fusionprotein being a transit peptide, which governs translocation of thepolypeptide. Especially preferred are chloroplast-specific transitpeptides which are cleaved enzymatically from the herbicide-bindingpolypeptide moiety after the herbicide-binding polypeptide has beentranslocated into the plant's chloroplasts. Particularly preferred isthe transit peptide derived from plastid transketolase (TK) or afunctional equivalent of this transit peptide (for example the transitpeptide of the small subunit of Rubisco or ferredoxin NADPoxidoreductase).

The polypeptide DNA or polypeptide cDNA required for the production ofexpression cassettes according to the invention is preferably amplifiedwith the aid of polymerase chain reaction (PCR). DNA amplificationmethods using PCR are known, for example from Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press (1990).The PCR-produced DNA fragments can expediently be checked by sequenceanalysis to avoid polymerase errors in constructs to be expressed.

The nucleotide sequence inserted, which encodes a herbicide-bindingpolypeptide, can be generated synthetically or obtained naturally orcomprise a mixture of synthetic and natural DNA components. In general,synthetic nucleotide sequences with codons which are preferred by plantsare prepared. These codons which are preferred by plants can bedetermined from codons whose proteins are most frequent and which areexpressed in most of the interesting plant species. When preparing anexpression cassette, various DNA fragments can be manipulated so as toobtain a nucleotide sequence which expediently reads in the correctsense and which is equipped with a correct reading frame. To connect theDNA fragments to each other, adaptors or linkers can be added to thefragments.

The promoter and terminator regions according to the invention shouldexpediently be provided, in the sense of the transcription, with alinker or polylinker comprising one or more restriction sites forinsertion of this sequence. As a rule, the linker has 1 to 10, usually 1to 8, preferably 2 to 6, restriction sites. Within the regulatoryregions, the linker generally has a size of less than 100 bp, frequentlyless than 60 bp, but at least 5 bp. The promoter according to theinvention can be either native or homologous or else foreign orheterologous to the host plant. The expression cassette according to theinvention comprises, in the 5′-3′-sense of transcription, the promoteraccording to the invention, any desired sequence and a region fortranscriptional termination. Various termination regions are mutuallyexchangeable as desired.

Furthermore, manipulations which provide suitable restriction sites orwhich remove excess DNA or restriction sites can be employed. Whereinsertions, deletions or substitutions, for example transitions andtransversions, are possible, in-vitro mutagenesis, “primer repair”,restriction or ligation may be used. In the case of suitablemanipulations such as restriction, “chewing-back” or filling upprojections for “blunt ends”, complementary ends of the fragments may beprovided for ligation purposes.

Especially important for the success according to the invention is theattachment of the specific ER retention signal SEKDEL (Schuoten, A. etal. Plant Mol. Biol. 30 (1996), 781-792), with which the averageexpression level is trebled to quadrupled. Other retention signals whichoccur naturally in plant and animal proteins which are localized in theER may also be used for constructing the cassette.

Preferred polyadenylation signals are plant polyadenylation signals,preferably those which correspond essentially to T-DNA polyadenylationsignals from Agrobacterium tumefaciens, in particular gene 3 of theT-DNA (octopin synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBOJ. 3 (1984) 835 et seq.) or functional equivalents.

An expression cassette according to the invention may comprise, forexample, a constitutive promotor (preferably the CaMV 35 S promotor),the LeB4 signal peptide, the gene to be expressed and the ER retentionsignal. The construction of the cassette is shown as a diagram in FIG. 2with reference to a single-chain antibody (scFv gene). The amino-acidsequence KDEL (lysine, aspartic acid, glutamic acid, leucine) ispreferably used as ER retention signal.

The fused expression cassette which encodes a polypeptide withherbicide-binding properties is preferably cloned into a vector, forexample pBin19, which is suitable for transforming Agrobacteriumtumefaciens. Agrobacteria which are transformed with such a vector canthen be used in the known manner for transforming plants, in particularcrop plants, eg. tobacco plants, by, for example, bathing wounded leavesor leaf sections in an Agrobacterial solution and subsequently growingthem in suitable media. The transformation of plants by means ofAgrobacteria is known, inter alia, from F. F. White, Vectors for GeneTransfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering andUtilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp.15-38, and from S. B. Gelvin, Molecular Genetics of T-DNA Transfer fromAgrobacterium to Plants, also in Transgenic Plants, pp. 49-78.Transgenic plants can be regenerated from the transformed cells of thewounded leaves or leaf sections in the known manner, and thesetransgenic plants contain a gene for the expression of a polypeptidewith herbicide-binding properties, integrated into the expressioncassette according to the invention.

To transform a host plant with a DNA encoding a herbicide-bindingpolypeptide, an expression cassette according to the invention isincorporated, as an insertion, into a recombinant vector whose vectorDNA contains additional functional regulation signals, for examplesequences for replication or integration. Suitable vectors aredescribed, inter alia, in “Methods in Plant Molecular Biology andBiotechnology” (CRC Press), (1993) chapter 6/7, pp.71-119.

Using the above-cited recombination and cloning techniques, theexpression cassettes according to the invention can be cloned intosuitable vectors which allow them to be multiplied, for example in E.coli. Suitable cloning vectors are, inter alia, pBR332, pUC series,M13mp series and pACYC184. Especially suitable are binary vectors whichcan replicate in both E. coli and agrobacteria, for example pBin19(Bevan et al. (1980) Nucl. Acids Res. 12, 8711).

The invention furthermore relates to the use of an expression cassetteaccording to the invention for the transformation of plants, plantcells, plant tissues or plant organs. The preferred aim upon use is themediation of resistance to plant-enzyme inhibitors.

Depending on the choice of the promoter, expression can take placespecifically in the leaves, in the seeds or in other plant organs. Suchtransgenic plants, their propagation material and their plant cells,plant tissues or plant organs are a further subject of the presentinvention.

The transfer of foreign genes into the genome of a plant is termedtransformation. In this process, the above-described methods oftransforming and regenerating plants from plant tissues or plant cellsare utilized for transient or stable transformation. Suitable methodsare protoplast transformation by polyethylene glycol-induced DNA uptake,the biolistic [sic] approach using the gene gun, electroporation,incubation of dry embryos in DNA-containing solution, microinjection andAgrobacterium-mediated gene transfer. The methods mentioned aredescribed, for example, in B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,editors: S. D. Kung and R. Wu, Academic Press (1993) 128-143 and inPotrykus, Annu.Rev.Plant Physiol.Plant Molec.Biol. 42 (1991) 205-225).The construct to be expressed is preferably cloned into a vector whichis suitable for the transformation of Agrobacterium tumefaciens, forexample pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).

Agrobacteria which have been transformed with an expression cassetteaccording to the invention can then be used in the known manner fortransforming plants, in particular crop plants such as cereals, maize,soya, rice, cotton, sugar beet, canola, sunflower, flax, potato,tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree,nut and Vitis species, for example by bathing wounded leaves or leafsections in an agrobacterial solution and subsequently growing them insuitable media.

Functionally equivalent sequences which encode a herbicide-bindingpolypeptide are, in accordance with the invention, those sequences whichstill have the desired functions, despite a different nucleotidesequence. Thus, functional equivalents encompass naturally occurringvariants of the sequences described herein, and also artificialnucleotide sequences, for example artificial nucleotide sequences whichhave been obtained by chemical synthesis and are adapted to the codonusage of a plant.

In particular, functional equivalent is to be understood as a natural orartificial mutation of an originally isolated sequence which encodes theherbicide-binding polypeptide, which mutation continues to show thedesired function. Mutations encompass substitutions, additions,deletions, exchanges or insertions of one or more nucleotide residues.Thus, the present invention also encompasses those nucleotide sequenceswhich are obtained by modifying this nucleotide sequence. The purpose ofsuch a modification can be, for example, the further limitation of theencoding sequence contained therein, or else, for example, the insertionof more cleavage sites for restriction enzymes.

Other functional equivalents are those variants whose function is lessor more pronounced, in comparison with the starting gene or genefragment.

Moreover, artificial DNA sequences are suitable as long as they inducethe desired resistance to herbicides, as described above. Suchartificial DNA sequences can be identified, for example, bybacktranslating proteins which have herbicide-binding activity and whichhave been constructed by means of molecular modeling, or by in vitroselection. Especially suitable are encoding DNA sequences which havebeen obtained by backtranslating a polypeptide sequence in accordancewith the codon utilization which is specific to the host plant. Thespecific codon utilization can be determined readily by an expertfamiliar with methods of plant genetics by computer-aided evaluation ofother, known genes of the plant to be transformed.

Further suitable equivalent nucleic acid sequences according to theinvention which must be mentioned are sequences which encode fusionproteins, where part of the fusion protein is a non-plant-derivedherbicide-binding polypeptide or a functionally equivalent part thereof.For example, the second part of the fusion protein can be a furtherpolypeptide with enzymatic activity, or an antigenic polypeptidesequence with the aid of which detection of scFvs expression is possible(for example myc-tag or his-tag). However, it is preferably a regulatoryprotein sequence, for example a signal or transit peptide, which directsthe polypeptide with herbicide-binding properties to the desired site ofaction.

However, the invention also relates to the expression products producedin accordance with the invention and to fusion proteins of a transitpeptide and a polypeptide with herbicide-binding properties.

Resistance/tolerance means, for the purposes of the present invention,the artificially acquired ability of plants to withstand the action ofplant enzyme inhibitors. It embraces the partial and, in particular,complete insensitivity to these inhibitors for the duration of at leastone plant generation.

The primary site of action of herbicides is generally the leaf tissue,so that leaf-specific expression of the exogenous herbicide-bindingpolypeptide is capable of providing sufficient protection. However, onewill understand readily that the action of a herbicide need not berestricted to the leaf tissue, but may also be effected in all remainingorgans of the plant in a tissue-specific manner.

In addition, constitutive expression of the exogenous herbicide-bindingpolypeptide is advantageous. On the other hand, inducible expression mayalso be desirable.

The efficacy of the transgenically expressed polypeptide withherbicide-binding properties can be determined for example in vitro byshoot meristem propagation on herbicide-containing medium in series withstaggered concentrations, or via seed germination tests. In addition,the herbicide tolerance, of a test plant, which has been altered withregard to type and level can be tested in greenhouse experiments.

The invention furthermore relates to transgenic plants, transformed withan expression cassette according to the invention, and to transgeniccells, tissues, organs and propagation material of such plants.Especially preferred are transgenic crop plants, for example cereals,maize, soya, rice, cotton, sugar beet, canola, sunflower, flax, potato,tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree,nut and Vitis species.

The transgenic plants, plant cells, plant tissues or plant organs can betreated with an active ingredient which inhibits the plant enzymes,whereby the plants, plant cells, plant tissues or plant organs whichhave not been transformed successfully die. Examples of suitable activeingredients are in particular5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid(acifluorfen) and 7-chloro-3-methylquinolin-8-carboxylic acid(quinmerac), and metabolites and functional derivatives of thesecompounds. The DNA which encodes a polypeptide with herbicide-bindingproperties and which has been inserted into the expression cassettesaccording to the invention can thus also be used as selection marker.

The present invention has the advantage, in particular in the case ofcrop plants, that, once a selected resistance of the crop plant to theplant enzyme inhibitors has been induced, such inhibitors can beemployed as specific herbicides to the non-resistant plants. Herbicidalcompounds from the groups bl-641 may be mentioned as examples of suchinhibitors, but not by way of limitation:

b1 1,3,4-Thiadiazoles:

buthidazole, cyprazole

b2 Amides:

allidochlor (CDAA), benzoylprop-ethyl, bromobutide, chlorthiamid,dimepiperate, dimethenamid, diphenamid, etobenzanid (benzchlomet),flamprop-methyl, fosamin, isoxaben, monalide, naptalame, pronamid(propyzamid), propanil

b3 Aminophosphoric acids:

bilanafos, (bialaphos), buminafos, glufosinate-ammonium, glyphosate,sulfosate

b4 Aminotriazoles:

amitrole

b5 Anilides:

anilofos, mefenacet

b6 Aryloxyalkanoic acids:

2,4-D, 2,4-DB, clomeprop, dichlorprop, dichlorprop-P, dichlorprop-P(2,4-DP-P), fenoprop (2,4,5-TP), fluoroxypyr, MCPA, MCPB, mecoprop,mecoprop-P, napropamide, napropanilide, triclopyr

b7 Benzoic acids:

chloramben, dicamba

b8 Benzothiadiazinones:

bentazone

b9 Bleachers:

clomazone (dimethazone), diflufenican, fluorochloridone, flupoxam,fluridone, pyrazolate, sulcotrione (chlormesulone)

b10 Carbamates:

asulam, barban, butylate, carbetamid, chlorbufam, chlorpropham,cycloate, desmedipham, di-allate, EPTC, esprocarb, molinate, orbencarb,pebulate, phenisopham, phenmedipham, propham, prosulfocarb,pyributicarb, sulf-allate (CDEC), terbucarb, thiobencarb (benthiocarb),tiocarbazil, tri-allate, vernolate

b11 Quinoline acids:

quinclorac, quinmerac

b12 Chloroacetanilides:

acetochlor, alachlor, butachlor, butenachlor, diethatylethyl,dimethachlor, metazachlor, metolachlor, pretilachlor, propachlor,prynachlor, terbuchlor, thenylchlor, xylachlor

b13 Cyclohexenones:

alloxydim, caloxydim, clethodim, cloproxydim, cycloxydim, sethoxydim,tralkoxydim,2-{1-[2-(4-chlorophenoxy)propyloxyimino]butyl}-3-hydroxy-5-(2H-tetrahydrothiopyran-3-yl)-2-cyclohexen-1-one

b14 Dichloropropionic acids:

dalapon

b15 Dihydrobenzofurans:

ethofumesate

b16 Dihydrofuran-3-ones:

flurtamone

b17 Dinitroanilines:

benefin, butralin, dinitramin, ethalfluralin, fluchloralin, isopropalin,nitralin, oryzalin, pendimethalin, prodiamine, profluralin, trifluralin

b18 Dinitrophenols:

bromofenoxim, dinoseb, dinoseb-acetate, dinoterb, DNOC

b19 Diphenyl ethers:

acifluorfen-sodium, aclonifen, bifenox, chlornitrofen (CNP),difenoxuron, ethoxyfen, fluorodifen, fluoroglycofen-ethyl, fomesafen,furyloxyfen, lactofen, nitrofen, nitrofluorfen, oxyfluorfen

b20 Dipyridylenes:

cyperquat, difenzoquat-methylsulfate, diquat, paraquat dichlorid

b21 Ureas:

benzthiazuron, buturon, chlorbromuron, chloroxuron, chlortoluron,cumyluron, dibenzyluron, cycluron, dimefuron, diuron, dymrone,ethidimuron, fenuron, fluormeturon, isoproturon, isouron, karbutilate,linuron, methabenzthiazuron, metobenzuron, metoxuron, monolinuron,monuron, neburon, siduron, tebuthiuron, trimeturon

b22 Imidazoles:

isocarbamid

b23 Imidazolinones:

imazamethapyr, imazapyr, imazaquin, imazethabenz-methyl (imazame),imazethapyr

b24 Oxadiazoles:

methazole, oxadiargyl, oxadiazon

b25 Oxiranes:

tridiphane

b26 Phenols:

bromoxynil, ioxynil

b27 Phenoxyphenoxypropionic esters:

clodinafop, cyhalofop-butyl, diclofop-methyl, fenoxapropethyl,fenoxaprop-p-ethyl, fenthiapropethyl, fluazifop-butyl,fluazifop-p-butyl, haloxyfop-ethoxyethyl, haloxyfop-methyl,haloxyfop-p-methyl, isoxapyrifop, propaquizafop, quizalofopethyl,quizalofop-p-ethyl, quizalofop-tefuryl

b28 Phenylacetic acids:

chlorfenac (fenac)

b29 Phenylpropionic acids:

chlorophenprop-methyl

b30 Protoporphyrinogen IX oxydase inhibitors:

benzofenap, cinidon-ethyl, flumiclorac-pentyl, flumioxazin, flumipropyn,flupropacil, fluthiacet-methyl, pyrazoxyfen, sulfentrazone, thidiazimin

b31 Pyrazoles:

nipyraclofen

b32 Pyridazines:

chloridazon, maleic hydrazide, norflurazon, pyridate

b33 Pyridinecarboxylic acids:

clopyralid, dithiopyr, picloram, thiazopyr

b34 Pyrimidyl ethers:

pyrithiobac-acid, pyrithiobac-sodium, KIH-2023, KIH-6127

b35 Sulfonamides:

flumetsulam, metosulam

b36 Sulfonylureas:

amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuronethyl,chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl,ethoxysulfuron, flazasulfuron, halosulfuronmethyl, imazosulfuron,metsulfuron-methyl, nicosulfuron, primisulfuron, prosulfuron,pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl,thifensulfuron-methyl, triasulfuron, tribenuron-methyl,triflusulfuron-methyl

b37 Triazines:

ametryn, atrazine, aziprotryn, cyanazine, cyprazine, desmetryn,dimethamethryn, dipropetryn, eglinazine-ethyl, hexazinone, procyazine,prometon, prometryn, propazine, secbumeton, simazine, simetryn,terbumeton, terbutryn, terbutylazine, trietazine

b38 Triazinones:

ethiozin, metamitron, metribuzin

b39 Triazolecarboxamides:

triazofenamid

b40 Uraciles

bromacil, lenacil, terbacil

b4Others:

benazolin, benfuresate, bensulide, benzofluor, butamifos, cafenstrole,chlorthal-dimethyl (DCPA), cinmethylin, dichlobenil, endothall,fluorbentranil, mefluidide, perfluidone, piperophos

The spectrum of action of functionally equivalent derivatives of plantenzyme inhibitors is comparable with the spectrum of action of thesubstances named individually, while the inhibitory activity (forexample expressed in g of inhibitor per hectare under cultivation,required for completely suppressing the growth of non-resistent plants)is lower, identical or higher.

The invention is now illustrated by the examples which follow, but isnot limited thereto:

General Cloning Methods

The cloning steps carried out within the scope of the present invention,for example restriction cleavages, agarose gel electrophoresis,purification of DNA fragments, transfer of nucleic acids tonitrocellulose and nylon membranes, linkage of DNA fragments,transformation of E. coli cells, cultivation of bacteria, multiplicationof phages and sequence analysis of recombinant DNA were carried out asdescribed by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press;ISBN 0-87969-309-6).

The bacterial strains used hereinbelow (E. coli, XL-I Blue) wereobtained from Stratagene. The agrobacterial strain used for thetransformation of plants (Agrobacterium tumefaciens, C58C1 with plasmidpGV2260 or pGV3850kan) was described by Deblaere et al. (Nucl. AcidsRes. 13 (1985) 4777). Alternatively, the agrobacterial strain LBA4404(Clontech) or other suitable strains may also be used. The vectors pUC19(Yanish-Perron, Gene 33(1985), 103-119) pBluescript SK-(Stratagene),pGEM-T (Promega), pZerO (Invitrogen), pBin19 (Bevan et al., Nucl. AcidsRes. 12(1984) 8711-8720) and pBinAR (Höfgen and Willmitzer, PlantScience 66 (1990) 221-230) were employed for cloning purposes.

Sequence Analysis of Recombinant DNA

Recombinant DNA molecules were sequenced using a laser fluorescence DNAsequencing apparatus from Pharmacia, using the method of Sanger (Sangeret al., Proc. Natl. Acad. Sci. USA 74(1977), 5463-5467).

Generation of Plant Expression Cassettes

A 35S CaMV promoter was inserted into plasmid pBin19(Bevan et al., Nucl.Acids Res. 12, 8711 (1984)) in the form of an EcoRI-KpnI fragment(corresponding to nucleotides 6909-7437 of the cauliflower mosaic virus(Franck et al. Cell 21 (1980) 285). The polyadenylation signal of gene 3of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984)835), nucleotides 11749-11939, was isolated in the form of aPvuII-HindIII fragment and, after SphI linkers had been added, clonedinto the PvuII cleavage site between the SphI-HindIII cleavage site ofthe vector. This gave plasmid pBinAR (Höfgen and Willmitzer, PlantScience 66 (1990) 221-230).

USE EXAMPLES Example 1

Since herbicides are not immunogenic, they must be coupled to a carriermaterial, for example KLH. If the molecule contains a reactive group,coupling may be effected directly; if not, a functional group isintroduced when the herbicide is synthesized or a reactive precursor isselected during synthesis so as to couple these molecules to the carriermolecule in a simple reaction step. Examples of coupling reactions canbe found in Miroslavic Ferencik in “Handbook of Immunochemistry”, 1993,Chapman & Hall, in the chapter Antigens, pages 20-49.

Repeated injection of this modified carrier molecule (antigen) is usedfor immunizing, for example, Balb/c mice. Once a sufficient number ofantibodies with binding to the antigen is detectable in the ELISA(enzyme-linked immunosorbent assay), the spleen cells of these animalsare removed and fused with myeloma cells in order to cultivate hybrids.“Herbicide-modified BSA” is additionally used as antigen in the ELISA soas to differentiate the immune response directed against the hapten fromthe KLH response.

Monoclonal antibodies are prepared by methods similar to known methods,for example as described in “Practical Immunology”, Leslie Hudson andFrank Hay, Blackwell Scientific Publications, 1989 or in “MonoclonalAntibodies: Principles and Practice”, James Goding, 1983, AcademicPress, Inc., or in “A practical guide to monoclonal antibodies”,J.Liddell and A. Cryer,1991, John Wiley& Sons; or Achim Möller and FranzEmling “Monoklonale Antikörper gegen TNF und deren Verwendung”[Monoclonal antibodies against TNF, and their use]. European PatentSpecification EP-A260610.

Example 2

The starting point of the investigation was a monoclonal antibody whichspecifically recognizes the herbicide quinmerac and which, additionally,has a high binding affinity. The hydridoma cell line selected ischaracterized in that the secreted monoclonal antibodies which aredirected against the herbicide antigen quinmerac have a high affinityand the specific sequences of the immunoglobulins are available (Berek,C. et al., Nature 316, (1985) 412-418). This monoclonal antibody againstquinmerac was the starting point for the construction of thesingle-chain antibody fragment (scFv-antiquinmerac).

First, mRNA was isolated from the hybridoma cells and transcribed intocDNA. This cDNA acted as a template for the amplification of thevariable immunglobulin genes VH and VK with the specific primers VH1BACK and VH FOR-2 for the heavy chain and VK2 BACK and MJK5 FON X forthe light chain (Clackson et al., Nature 352, (1991) 624-628). Thevariable immunoglobulins isolated were the starting point for theconstruction of a single-chain antibody fragment (scFv-antiquinmerac).In the subsequent fusion PCR, three components VH,VK and a linkerfragment were combined in a PCR reaction, and the scFv-antiquinmerac wasamplified (FIG. 3).

Functional characterization (antigen binding activity) of thescFv-antiquinmerac gene constructed was carried out after expression ina bacterial system. To this end, the scFv-antiquinmerac was synthesizedin E. coli as a soluble antibody fragment, using the method ofHoogenboom, H. R. et al., Nucleic Acids Research, 19, (1991) 4133-4137.Activity and specificity of the antibody fragment constructed werechecked in an ELISA assay (FIG. 4).

To allow seed-specific expression of the antibody fragment in tobacco,the scFv-antiquinmerac gene was cloned downstream from the LeB4promoter. The LeB4 promoter, which had been isolated from Vicia faba,shows strictly seed-specific expression of various foreign genes intobacco (Bäumlein, H. et al., Mol. Gen. Genet. 225, (1991) 121-128).Transport of the scFv-antiquinmerac polypeptide into the endoplasmaticreticulum resulted in stable accumulation of large amounts of antibodyfragment. To this end, the scFv-antiquinmerac gene was fused with asignal peptide sequence which guarantees entry into the endoplasmaticreticulum and with the ER retention signal SEKDEL, which guarantees thatthe polypeptide remains in the ER (Wandelt et al.,1992) (FIG. 5).

The expression cassette constructed was cloned into the binary vectorpGSGLUC 1 (Saito et al., 1990) and transferred into the agrobacteriumstrain EHA 101 by electroporation. Recombinant agrobacterial clones wereused for the subsequent transformation of Nicotiana tabacum. 70-140tobacco plants were regenerated per construct. Seeds in differentdevelopmental stages were harvested from the regenerated transgenictobacco plants, following self-pollination. The soluble proteins wereobtained from these seeds in an aqueous buffer system, after extraction.Analysis of the transgenic plants demonstrates that fusion of thescFv-antiquinmerac gene to the DNA sequence of the ER retention signalSEKDEL allowed a maximum accumulation of 1.9% scFv-antiquinmerac proteinto be obtained in the mature seed.

The scFv-antiquinmerac gene constructed had a size of approximately 735bp. The variable domains were fused to each other in the sequenceVH-L-VL.

The specific selectivity was determined in the extracts of the maturetobacco seeds using a direct ELISA. The values obtained demonstrateclearly that the protein extracts contain functionally active antibodyfragments.

Example 3

Seed-specific expression and concentration of single-chain antibodyfragments in the endoplasmatic reticulum of cells of transgenic tobaccoseeds, under the control of the USP promoter.

Starting point of the investigations was a single-chain antibodyfragment against the herbicide quinmerac (scFv-antiquinmerac). Thefunctional characterization (antigen binding activity) of thisscFv-antiquinmerac gene constructed was carried out following expressionin a bacterial system and following expression in tobacco leaves.Activity and specificity of the antibody fragment constructed werechecked with ELISA assays.

To allow seed-specific expression of the antibody fragment in tobacco,the scFv-antiquinmerac gene was cloned downstream from the USP promoter.The USP promoter, which had been isolated from Vicia faba, showsstrictly seed-specific expression of various foreign genes in tobacco(Fiedler, U. et al., Plant Mol. Biol. 22, (1993) 669-679). Transport ofthe scFv-antiquinmerac polypeptide into the endoplasmatic reticulumresulted in stable accumulation of large amounts of antibody fragment.To this end, the scFv-antiquinmerac gene was fused with a signal peptidesequence which guarantees entry into the endoplasmatic reticulum andwith the ER retention signal SEKDEL, which guarantees that thepolypeptide remains in the ER (Wandelt et al., 1992) (FIG. 1).

The expression cassette constructed was cloned into the binary vectorpGSGLUC 1 (Saito et al., 1990) and transferred into the Agrobacteriumstrain EHA 101 by electroporation. Recombinant agrobacterial clones wereused for the subsequent transformation of Nicotiana tabacum. Seeds indifferent developmental stages were harvested from the regeneratedtransgenic tobacco plants, following self-pollination. The solubleproteins were obtained from these seeds in an aqueous buffer system,after extraction. Analysis of the transgenic plants demonstrates thatfusion of the scFv-antiacifluorfen [sic] gene to the DNA sequence of theER retention signal SEKDEL under the control of the USP promoter causedsingle-chain antibody fragments with a binding affinity for quinmerac tobe synthesized as early as day 10 of the seed development.

Example 4

To achieve ubiquitous expression of the antibody fragment in the plant,especially in leaves, the scFv-antiquinmerac gene was cloned downstreamof the CaMV 35 S promoter. This strong constitutive promoter mediatesexpression of foreign genes in virtually all plant tissues (Benfey andChua, Science 250 (1990), 956-966. Transport of the scFv-antiquinmeracprotein into the endoplasmatic reticulum allowed stable accumulation oflarge amounts of antibody fragment to be obtained in the leaf material.First, the scFv-antiquinmerac gene was fused to a signal peptidesequence which ensures entry into the endoplasmatic reticulum and to theER retention signal KDEL, which ensures that the product remains in theER (Wandelt et al., Plant J. 2(1992), 181-192). The expression cassetteconstructed was cloned into the binary vector pGSGLUC 1 (Saito et al.,Plant Cell Rep. 8(1990), 718-721) and transferred into the Agrobacteriumstrain EHA 101 by electroporation. Recombinant agrobacterial clones wereused for the subsequent transformation of Nicotiana tabacum.Approximately 100 tobacco plants were regenerated. Leaf material ofvarious developmental stages was removed from the regenerated transgenictobacco plants. The soluble proteins were obtained from this leafmaterial in an aqueous buffer system, following extraction. Subsequentanalyses (western blot analyses and ELISA assays) demonstrated that amaximum accumulation of more than 2% of biologically activeantigen-binding scFv-antiquinmerac polypeptide was obtained in theleaves. The high expression values were determined in fully grown greenleaves, but the antibody fragment was also detected in senescent leafmaterial.

Example 5

PCR amplification of a fragment of the cDNA encoding the single-chainantibody against acifluorfen and quinmerac with the aid of syntheticoligonucleotides.

The PCR amplification of the single-chain antibody cDNA was carried outin a DNA thermal cycler from Perkin Elmer. The reaction mixturescontained 8 ng/μl single-stranded template cDNA, 0.5 μM of the relevantoligonucleotides, 200 μM nucleotides (Pharmacia), 50 mM KCl, 10 mMTris-HCl (pH 8.3 at 25° C., 1.5 mM MgCl₂) and 0.02 U/μl Taq polymerase(Perkin Elmer). The amplification conditions were set as follows:

Annealing temperature: 45° C. Denaturation temperature: 94° C.,Elongation temperature: 72° C., Number of cycles: 40

The result is a fragment of approx. 735 base pairs, which was ligatedinto the vector pBluescript. The ligation mixture was used fortransforming E. coli XL-I Blue, and the plasmid was amplified. Regardinguse and optimization of polymerase chain reaction, see: Innis et al.,1990, PCR Protocols, A Guide to Methods and Applications, AcademicPress.

Example 6

Production of transgenic tobacco plants which express a cDNA encoding asingle-chain antibody with herbicide-binding properties.

Plasmid pGSGLUC 1 was transformed into Agrobacterium tumefaciensC58C1:pGV2260. To transform tobacco plants (Nicotiana tabacum cv. SamsunNN), a 1:50 dilution of an overnight culture of a positively transformedagrobacterial colony in Murashige-Skoog medium (Physiol. Plant. 15(1962) 473 et seq.) containing 2% of sucrose (2MS medium) was used. In aPetri dish, leaf disks of sterile plants (each approx. 1 cm²) wereincubated for 5-10 minutes in a 1:50 agrobacterial dilution. This isfollowed by 2 days' incubation in the dark at 25° C. on 2MS mediumcontaining 0.8% Bacto-Agar. Cultivation was continued after 2 days in 16hours light/8 hours dark and continued in a weekly rhythm on MS mediumcontaining 500 mg/l Claforan (cefotaxim-sodium), 50 mg/l kanamycin, 1mg/l benzylaminopurine (BAP), 0.2 mg/l naphthylacetic acid and 1.6 g/lglucose. Growing shoots were transferred to MS medium containing 2%sucrose, 250 mg/l Claforan and 0.8% Bacto-Agar.

Example 7

Stable accumulation of the single-chain antibody fragment against theherbicide quinmerac in the endoplasmatic reticulum.

Starting point of the investigations was a single-chain antibodyfragment against the herbicide quinmerac(scFv-antiquinmerac) which isexpressed in tobacco plants. Quantity and activity of thescFv-antiquinmerac polypeptide synthesized were determined in westernblot analyses and ELISA assays.

To make possible expression of the scFv-antiquinmerac gene in theendoplasmatic reticulum, the foreign gene was expressed under thecontrol of the CaMV 53S promoter as a translation fusion with the LeB4signal peptide (N-terminal) and the ER retention signal KDEL(C-terminal). Transport of the scFv-antiquinmerac polypeptide into theendoplasmatic reticulum allowed stable accumulation of large quantitiesof active antibody fragment. After the leaf material had been harvested,sections were frozen at −20° C. (1), lyophilized (2) or dried at roomtemperature (3). The soluble proteins were obtained from the leafmaterial in question by extraction in an aqueous buffer, and thescFv-antiquinmerac polpypeptide was purified by affinity chromatography.Equal amounts of purified scFv-antiquinmerac polypeptide (frozen,lyophilized and dried) were employed for determining the activity of theantibody fragment (FIG. 6). FIG. 6A shows antigen binding activity ofthe scFv-antiquinmerac polypeptide from fresh (1), lyophilized (2) anddried (3) leaves. FIG. 6B shows the respective amounts ofscFv-antiquinmerac protein (approximately 100 ng), used for the ELISAanalyses, determined by western blot analyses. The sizes of the proteinmolecular weight standards are shown on the left. Approximatelyidentical antigen binding activities were found.

Example 8

To demonstrate the herbicide tolerance of the transgenic tobacco plantswhich produce a polypeptide with herbicide-binding properties, thesetobacco plants were treated with various amounts of acifluorfen andquinmerac. It was possible to demonstrate in all cases, in thegreenhouse, that the plants expressing an scFv-antiacifluorfen andscFv-antiquinmerac, respectively, showed tolerance to the herbicides inquestion in comparison with the control.

1 1 6 PRT Unknown Endoplasmic reticulum retention signal 1 Ser Glu LysAsp Glu Leu 1 5

We claim:
 1. A process for the production of a5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or7-chloro-3-methyllquinoline-8-carboxylic acid tolerant plant, saidprocess comprising the step of transforming plants to produce anexogenous 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or7-chloro-3-methylIquinoline-8-carboxylic-binding antibody in the plants,and selecting a tolerant plant.
 2. The process as claimed in claim 1,wherein the exogenous antibody is a single-chain antibody bindingfragment.
 3. The process as claimed in claim 1, wherein the exogenousantibody is a complete antibody or a binding fragment.
 4. The process asclaimed in claim 1, wherein the plant is mono- or dicotyledonous.
 5. Theprocess as claimed in claim 4, wherein the plant is tobacco.
 6. Theprocess as claimed in claim 1, wherein the antibody is expressedconstitutively in the plant.
 7. The process as claimed in claim 1,wherein expression of the exogenous antibody in the plant is induced. 8.The process as claimed in claim 1, wherein the exogenous antibody isexpressed in the leaves of the plant.
 9. The process as claimed in claim1, wherein the exogenous antibody is expressed in the seeds of theplant.
 10. An expression cassette for plants, comprising in operativelinkage a plant-functional promoter, a targeting sequence, a geneencoding an 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acidor 7-chloro-3-methyllquinoline-8-carboxylic acid binding antibody, and aterminator.
 11. The expression cassette as claimed in claim 10, whereinsaid promoter is the CaMV 35S promoter.
 12. The expression cassette asclaimed in claim 10, wherein said gene encodes a single-chain antibody.13. The expression cassette as claimed in claim 10, wherein the geneencoding a 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acidor 7-chloro-3-methyllquinoline-8-carboxylic acid-binding antibody is inthe form of a translation fusion with a gene encoding another functionalprotein or is employed as the gene to be expressed.
 14. The expressioncassette as claimed in claim 10, wherein an antibody gene to beexpressed is isolated from a hybridoma cell or phage display libraries.15. A process of transforming dicotyledonous or monocotyledonous plantscomprising the step of introducing the expression cassette as claimed inclaim 10 into the plant genome, wherein the5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or7-chloro-3-methyllquinoline-8-carboxylic acid-binding antibody isexpressed seed- or leaf specifically.
 16. The process as claimed inclaim 15, wherein the expression cassette is first transferred into abacterial strain and resulting recombinant transformed bacterial clonesare used for the transformation of the dicotyledonous ormonocotyledonous plants to express a5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or7-chloro-3-methyllquinoline-8-carboxylic-binding antibody seed- or leaf-specifically.
 17. A process for the transformation of a plant, the saidprocess comprising the step of introducing a gene sequence which encodesa 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or7-chloro-3-methyllquinoline-8-carboxylic acid-binding antibody into aplant cell, into callus tissue, into an entire plant or intoprotoplasts.
 18. The process as claimed in claim 17, whereinintroduction is performed by agrobacterium.
 19. The process as claimedin claim 17, wherein introduction is performed by electroporation. 20.The process as claimed in claim 17, wherein introduction is by aparticle bombardment method.
 21. A method of producing a5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or7-chloro-3-methylIquinoline-8-carboxylic acid-binding antibody, saidmethod comprising the step of introducing into a plant genome andexpressing a gene which encodes said antibody in a plant or cells of aplant, and subsequently isolating the antibody.
 22. A plant comprisingthe expression cassette as claimed in claim 10, wherein the expressioncassette imparts tolerance to5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid or7-chloro-3-methyllquinoline-8-carboxylic acid.
 23. The process asclaimed claim 18, wherein the agrobacterium is Agrobacteium tumefaciens.24. A process for selecting transformed plant cells, said processcomprising the step of transforming cells with the expression cassetteas claimed in claim 10, and culturing said transformed plant cells inthe presence of herbicides.